Clinically relevant atovaquone-resistant human malaria parasites fail to transmit by mosquito

Long-acting injectable medications, such as atovaquone, offer the prospect of a “chemical vaccine” for malaria, combining drug efficacy with vaccine durability. However, selection and transmission of drug-resistant parasites is of concern. Laboratory studies have indicated that atovaquone resistance disadvantages parasites in mosquitoes, but lack of data on clinically relevant Plasmodium falciparum has hampered integration of these variable findings into drug development decisions. Here we generate atovaquone-resistant parasites that differ from wild type parent by only a Y268S mutation in cytochrome b, a modification associated with atovaquone treatment failure in humans. Relative to wild type, Y268S parasites evidence multiple defects, most marked in their development in mosquitoes, whether from Southeast Asia (Anopheles stephensi) or Africa (An. gambiae). Growth of asexual Y268S P. falciparum in human red cells is impaired, but parasite loss in the mosquito is progressive, from reduced gametocyte exflagellation, to smaller number and size of oocysts, and finally to absence of sporozoites. The Y268S mutant fails to transmit from mosquitoes to mice engrafted with human liver cells and erythrocytes. The severe-to-lethal fitness cost of clinically relevant atovaquone resistance to P. falciparum in the mosquito substantially lessens the likelihood of its transmission in the field.


Supplementary Figure
. In vitro selection of gametocyte-competent, atovaquoneresistant, P. falciparum.A Representative timeline for selecting atovaquone-resistant parasites from under constant drug pressure.B Compilation of thirteen attempted selections.NF54 parent line was known to generate infectious gametocytes; parasites from trials 10-13 were low passage isolates from clinical trial volunteers 1 .Total number of parasites pressured was 7.8 x 10 9 .C Sequence data revealing an A-to-C mutation resulting in Y268S change.No evidence for heteroplasmy was detected in any of the WT or mutant samples.In culture . Evaluation of ookinetes in anopheline mosquito midguts at 20-26 h after membrane feed.A Sum of mature and immature ookinetes in pooled samples obtained from ten An. stephensi midguts, in 60 independent 1000X microscopy fields.Data are from one biological experiment.B Sum of mature and immature ookinetes seen in ten An. gambiae midguts, in 60 independent 1000X microscopy fields.Data are from one biological experiment.C Immature WT control ookinete in An. stephensi.D Mature WT control ookinetes in An. stephensi.E Immature WT control ookinete in An. gambiae.F Mature WT control ookinetes in An. gambiae.Bars, 10 µm.Arrows, representative ookinetes.Source data are provided as a Source Data file. .An. stephensi salivary gland sporozoite loads over time for wild type or cytochrome b mutant P. falciparum.
. Mixed infection with WT and Y268S mutant P. falciparum in An. stephensi mosquitoes, from three biological replicate experiments.Parasites were fed to mosquitoes in three cohorts: WT control (0.5% gametocytemia), a 1:1 mixture of WT:Y268S (0.25% WT:0.25%Y268S gametocytemia), or a 1:10 mixture of WT:Y268S (0.05% WT:0.45%Y268S gametocytemia).Results are from three independent biological experiments.A Oocysts were counted at 7-9 d after membrane feed.Bars are median (values indicated); WT range 0 to 42, 1:1 range 0 to 23, 1:10 range 0 to 6; n, number of mosquitoes dissected.**P = 0.004, ****P < 0.0001 by Kruskal-Wallis.Pie charts are percent of mosquitoes infected; ****P <0.0001 by Chi-square.B Salivary glands were removed for sporozoite counts 14-16 d after infection.Total number of mosquitoes were 90 for WT, 91 for 1:1, and 90 for 1:10.Symbols, average sporozoites/mosquito for each experiment; bars, median (values indicated) and ranges (8,800 to 17,000 for WT; 3,900 to 9,500 for 1:1; 200 to 2,100 for 1:10).Overall Chi-square 0.039; not powered for further analysis.C Parasite numbers as a function of percent WT gametocytes in the bloodmeal.For oocysts y = 0.11x -0.14, R 2 0.998; for sporozoites y = 107x + 440, R 2 0.988.D RFLP analysis for Y268S mutant DNA in mosquito tissues, obtained in three independent . Evaluation of paired wild type and L144S P. falciparum in vitro and in An. stephensi mosquitoes.A Atovaquone cytotoxicity against WT or L144S asexual erythrocytic parasites (EC50 0.68 or 48 nM, respectively).Depicted are mean ± SD of quadruplicate determinations in each of three independent biological experiments (total n = 12; some SD are too small to extend outside the symbols); R 2 ≥0.997.Susceptibilities of WT and L144S mutant cells to artemisinin (9.8 versus 8.5 nM) or chloroquine (8.2 versus 8.8 nM) were not significantly different.B Male gametocyte exflagellation adjusted to 1.5% gametocytemia.Median numbers in two independent biological replicates are indicated (ranges 4 to 11 for WT, and 0 to 9 for mutant); n, total number of fields examined; *P = 0.0253, two-sided Mann-Whitney test, single comparison.C Oocysts in four independent biological replicates at 9-10 d after membrane feed.Median number of oocysts per midgut is indicated (ranges 0 to 76 for WT, and 0 to 55 for L144S); n, total number of mosquitoes dissected; ****P <0.0001, two-sided Mann-Whitney test, single comparison; pie charts percent of midguts infected, two-sided Fisher's exact test.D Photomicrograph of representative midgut sample from An. stephensi infected with L144S P. falciparum gametocytes.Bar, 100 µm; arrow, L144S oocyst.E P. falciparum sporozoite counts in four independent biological replicates at 17-20 d after membrane feed, entailing a total of 109 WT-fed and 118 mutant-fed mosquitoes.Symbols, for each experiment, average number of sporozoites per mosquito; bars, median number of sporozoites (indicated) and ranges 13,000 to 38,000 for WT; 3 to 22,000 for mutant); P = 0.1143, two-sided Mann-Whitney test, single comparison.Source data are provided as a Source Data file.
band of the expected size for the Stevor fragment.Full experimental details are provided in the legends for Figs.3b and c.Supplementry Figure S11.Determination of human red cell engraftment in huHepmice.Blood collected from mice was treated with no antibody or with APC-conjugated rat anti-mouse glycophorin monoclonal antibody prior to separation by flow cytometry.A Unstained control sample showing both human and mouse erythrocytes within the APCgate.B Similar analysis of blood incubated with antibody prior to cytometry distinguishes between human (APC-) and mouse (APC+) erythrocytes.

RFLP to detect WT and mutant parasites in mixed infections
a Residues in lower case red encode base changes to generate NsiI (a) or PstI (c) endonuclease sites that distinguish mutant from WT at codon 268.

Supplementary Figure S2. PCR primers used in these studies. A Primer sequences and
descriptions.B Graphic of primers for Pfcytb sequencing.C Graphic of primers for Pfcytb nested PCR.D Graphic of primers for RFLP analysis.To identify low levels of Y268S-encoding DNA in mosquito tissues, an RFLP method 2 was modified as follows.

PfCytb SEQUENCING PfCytb NESTED PCR B C RFLP ANALYSIS D DNA
was isolated from midguts and salivary glands (Monarch Genomic DNA purification kit, NEB), and about 10 ng DNA was analyzed.In the primary PCR, a 939 bp product containing the mutation site was amplified with Taq DNA polymerase (NEB).Then 250 bp nested products were amplified by Taq DNA polymerase from 1 µL primary reaction product plus primer pairs: either CytB8 and CytB5 for detection of WT Y268, or CtyB8 and CytB9 for detection of mutant Y268S.Reverse primer CytB5, in conjunction with WT sequence TAT in the template DNA, results in an NsiI recognition site (ATGCAT) in the nested PCR product.Reverse primer CytB9, in conjunction with mutant sequence TCT in the template, generates a PstI recognition sequence (CTGCAG).For RFLP analysis, 5 µL of amplified DNA was digested (37 °C, overnight) with 1U NsiI or PstI (NEB).Products were resolved in 3% agarose and visualized by GelStar (Lonza) fluorescence.Successful digestion by either NsiI or PstI yields 224 and 26 bp products.From mixed cultures, an allelic population as low as 2% (NsiI) or 10% (PstI) can be detected.Asynchronous parasites grown continuously over four months and without shaking were sampled for differential count at every 3-4 day passage.Depicted are percent of each stage in WT (left panel) and Y268S mutant (right panel) cultures.The tendency in WT cultures for trophozoites to be the least abundant form is lost in mutant populations.This alteration suggests that in mutants the maturation of metabolically active trophozoites is prolonged, a fitness cost consistent with impaired pyrimidine synthesis as a consequence of cytochrome b mutation.Source data are provided as a Source Data file.
Supplementary FigureS3.Growth characteristics in vitro of WT and cytochrome b Y268S asexual erythrocytic parasites, obtained from one biological replicate.A Cell count was monitored at indicated intervals for a culture seeded at 0.1% parasitemia and not passaged.Relative to WT, mutant cells had a prolonged lag phase, delayed and lower peak parasitemia, and did not persist in stationary phase.Each point is the average of two replicate counts.B Progeny within late schizonts were counted in SYBR Gold-stained thin smear by fluorescence microscopy at 1000x magnification.Indicated are median values (ranges 4 to 23 for WT, and 4 to 23 for Y268S); n = 100, ***P = 0.0001, two-sided Mann-Whitney test, single comparison.C

P. falciparum counts in tissues of An. gambiae, from three biological replicate experiments
. A Midguts were examined at 7-10 d after membrane feed.Median number of oocysts per midgut is indicated; ranges 0 to 131 for WT, or 0 to 2 for Y268S; n, total number of mosquitoes dissected; ****P <0.0001, two-sided Mann-Whitney test, single comparison; pie charts, percent of mosquitoes infected, ****P <0.0001, two-sided Fisher's exact test.B Salivary glands were dissected 14-19 d after membrane feed, from a total of 90 mosquitoes infected with WT or 100 infected with Y268S mutant parasites.Symbols, for each experiment, average number of sporozoites per mosquito.Bars, median values (indicated) and ranges (1700 to 6200 for WT; no sporozoites were seen in any Y268S sample); not powered for statistical analysis.Source data are provided as a Source Data file.

Representative experimental timeline of P. falciparum transmission from An. stephensi to huHep mice.
✱✱✱✱biological experiments, each assayed in two independent technical replicates.Upper panel, NsiI cleaves only the WT Pfcytb sequence, whereas PstI cleaves only the sequence encoding Y268S (lower panel).In both cases a 224 bp product is released, allowing detection of an allelic population as low as 2% (NsiI) or 10% (PstI) (Supplementary Fig.S2A,D).1, WT control; 2, WT:Y268S at 1:1; 3, WT:Y268S at 1:10; W, WT DNA as assay control; M, Y268S DNA as assay control.Source data are provided as a Source Data file.